JPH0684952B2 - Analytical method and sensor electrode - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is capable of detecting the presence of chemical sensors, particularly N-acetylaromatic primary amines, such as paracetamol, in whole blood and measuring its amount or monitoring its level. It relates to a chemical sensor.

Paracetamol (N-acetyl p-aminophenol)
It is widely used as an analgesic that acts quickly and generally has no side effects. Although this drug has efficacious effects and favorable interactions with other drugs, the availability of it has led to its increased use in suicide.

Lethal doses are lethal as a result of extensive liver necrosis.

Paracetamol is thought to act by blocking the synthesis of certain of the prostaglandins and lipopolysaccharides that are thought to be sensitive to pain receptors.

Excretion of this substance from the human body is considered to occur after conjugation in the liver. The current theory is that paracetamol conjugates at low concentrations with sulfate or glucuronide, but at higher concentrations oxidative metabolic pathways are formed to form cysteine, mercapturic acid conjugates and then with active intermediates and reduced glutathione. The reaction is carried out. It is believed that extremely high concentrations of paracetamol become depleted in hepatic diminished glutathione pools and the resulting high levels of activated intermediates cause hepatocyte necrosis. Furthermore, the active chemical group of the intermediate that causes cell damage is thought to be the sulfhydryl group.

The early clinical phase of liver necrosis is the only obvious symptom of paracetamol intoxication, which does not occur until a few hours 10 to 12 hours after ingestion. Furthermore, this poisoning must be treated before necrosis occurs and thus the first clinical signs. The usual treatment is gastric lavage or ingestion of adsorbents such as activated charcoal (if medical benefits are available shortly after ingestion), followed by administration of substances with sulfhydryl groups that competitively block the action of activated intermediates. including.

However, administration of sulfhydryl-containing antidotes is
After 10 hours or more, it was confirmed to be dangerous if the serum paracetamol concentration was not met correctly.

Therefore, it would be desirable to provide a method by which a rapid and accurate determination of paracetamol levels can be made, thereby enabling early establishment of a diagnostic and effective treatment.

Previous attempts to provide such methods have been based on chromatographic methods that require time, skill and expensive equipment. Alternatively, a more specific colorimetric method based on the detection of p-aminophenol (PAP) has been used. p-Aminophenol (MP186 ° C) is produced by enzymatic degradation of paracetamol, again requiring expensive equipment and skilled personnel. A particular limitation of the colorimetric method is the use of serum or plasma rather than whole blood due to red blood cell interference.

Therefore, it would be desirable to provide a method for the analysis of paracetamol which is performed by a relatively immature person without the use of large and expensive equipment. Such methods are preferably used in vitro with whole blood or in vivo.

European Patent Application No. 82305597 consists of a conductive material,
Disclosed and claimed is a sensor electrode having a combination of an enzyme on at least its outer surface and a mediator compound that transports electrons to the electrode when the enzyme is effective for the catalytic reaction.

The purpose of such electrodes is to detect the presence, measure and / or monitor the presence of one or more selected components capable of carrying out a reaction catalyzed by the enzyme.

The present invention is based on the finding that certain general reaction mechanisms are particularly useful as the basis for the analysis of paracetamol and related compounds.

In a first aspect of the invention there is provided an assay method of the type in which an electrode kept at a suitable potential is contacted with the following sample and a system containing the enzyme: a) a sample suspected of containing paracetamol or a derivative thereof, And b) an enzyme capable of catalyzing the hydrolysis of an N-acylated aromatic primary amine or derivative thereof, provided that the current flowing through the electrode is the amount of hydrolysis product formed. , Thus a measure of the concentration of N-acylated aromatic primary amine or derivative thereof in the sample.

It should be noted that this system does not use a mediator and in this respect differs from the applicant's earlier application.

The nature and manufacture of the sensor electrode is disclosed in the applicant's pending application, whose title is "analyzer and sensor electrode therefor". Such electrodes are preferred for the practice of the present invention, the electrodes being coated with an enzyme on their surface, but preferably without a mediator compound.

Therefore, a second aspect of the invention is a sensor electrode having an enzyme on the surface which is capable of catalyzing the hydrolysis of paracetamol or a derivative of paracetamol to the product, provided that the current flowing through the electrode is And the resulting concentration of paracetamol or its derivative at the surface.

Conveniently, the enzyme is of the type defined as EC 3.5.1.13 and is as an aryl acylamidase [IUB] (otherwise known as aryl acylamido aminohydrolase). It is named.

The enzyme is preferably obtained from bacteria.

Paracetamol was added to the p- for analysis on the electrode surface.
A large number of bacterial enzymes that convert directly to aminophenols have been studied.

The following reaction schemes have been postulated for the processes that occur in analytical systems using these aryl acylamidases: Under the action of bacterial aryl acylamidases, paracetamol hydrolyzes to produce p-aminophenol and acetic acid. If the potential difference between p-aminophenol in solution and the electrode surface is such that the low energy band at the electrode is found by the electron, then the electron will be accepted by the conduction band of the electrode. Therefore, when a potential difference is applied to the electrodes, electro-oxidation
ion) occurs with respect to the reference electrode.

In a particular example of the invention, oxygen is generated from Fusarium species.

In another particular embodiment of the invention, oxygen is added to Pseudomonas (Pseu).
domonas) seeds.

The invention will now be described by way of example with reference to the drawings.

Working electrodes of various materials were used: Gold, platinum, glassy carbon and nickel (solid) electrodes were made from electrode disks and brass tie rods housed in Teflon coatings. In the laboratory, graphite paste electrodes were made from bronze interconnects housed in araldite (obtained from Radio Spares). The Araldite coating (held in the glass collar) included a cup with a hole at one end to expose the connector. Graphite paste material was filled into this cup.

A graphite paste was made using graphite powder mixed with Nujol or graphite powder mixed with Araldite and cured in a cup. In either case, the counter electrode was held near the working electrode material so that electrons could easily flow.

The following example shows the results using a technique involving the present invention.

Reference Example 1 Cyclic voltammetry of P-aminophenol Potassium dihydrogen phosphate [5.31 g; British Drug Houses (Analar manufactured by BDH) and dipotassium hydrogen phosphate [13.92 g; Analer manufactured by BDH] More buffer solution was prepared. At this time, these were added to Milli-Q wate.
Dissolved in r), the pH was adjusted to 7 and the final volume was 1 liter.

Add p-aminophenol solution to approximately 80 ml of Milly Q water.
Adjusted by dissolving 4.56 mg of p-aminophenol (BDH) and adjusted to pH 11 with 0.1 M NaOH. The solution was then acidified to pH 9 with 0.1M HCl and the final volume was adjusted with Milly Q water.
It was 100 ml. The p-aminophenol solution was protected from light and used immediately after preparation.

The working electrode was made from a range of different materials such as gold, vitreous carbon and especially pyrolytic graphite. The electrode was periodically cleaned during the experiment using a slurry of 0.3 μ alumina (BDH) in water to remove oxidation products and impurities from the electrode surface. The alumina was then ultrasonically removed from the electrode surface.

The cyclic voltammogram is displayed on a saturated calomel electrode (SC
For E) the potential was obtained from a range of solutions by sweeping between zero and +400 mV and -200 mV. Applied potential is 50 mV
It was controlled by a potentiostat (Jtron, AS Scientific Hook Abington) using an operating speed of / s.

The oxidation current generated was recorded with a Gludocilese 60000 chart recorder. The X-axis shows the applied potential and the Y-axis shows the generated current. A cyclic voltammogram of p-aminophenol (at a final concentration of 1 mM) is shown in FIG.

Example 1 Sensor incorporating aryl acylamidase Paracetamol (N-acetyl p-aminophenol;
Sigma Chemical Co.) was dissolved in potassium phosphate buffer to give a final concentration of 25 mM. Aryl acylamidases extracted from Pseudomonas species were lyophilized and supplied in 10 ml glass bottles by PHLS Center for Applied Microbiology End Research, Porton Down, Salisbury. The enzyme was stored at -20 ° C and the aryl acylamidase extracted from Fusarium sp. Was supplied by Sigma Chemical Co.
Reproduced with l Millie Q water.

The enzyme solution was analyzed periodically using the method of Atkinson A. Hammond, P.M.Blythe C.P. and Skawen M.D. (British Patent No. 2089978B). In this system, 0.1% containing 25 ml of 0.2% (W / V) aqueous solution of anhydrous copper sulfate mixed with 0.4 ml of 0.0880 ammonia.
(W / V) Orthocresol aqueous solution 1 ml and ammoniacal copper sulfate 0.1 ml were added to water 1.4 ml in a disposable cuvette. This solution was mixed well and 0.5 ml of standard p-aminophenol solution was added. The solution was then remixed and allowed to sit for 5 minutes before measuring the absorption of the solution at 615 nm. One unit of enzyme was defined as the conversion of 1 μmol paracetamol to para-aminophenol per minute at pH 7 and temperature 37 ° C.

The electrodes used are the same as those described in the above reference example.

For cyclic voltammograms, cells were loaded with 24 μl of paracetamol solution (25 mM as above) and 576 μl of buffer solution.

Cyclic voltammograms were recorded both in the presence and absence of 0.9 U arylacyl amidase (120 μl of the enzyme solution above). To ensure that the reaction started, each sample was incubated at 37 ° C for 2 minutes before starting the scan.

The cyclic voltammogram is shown in FIG. FIG. 3 shows that substantial changes were observed in the voltammogram contour upon addition of the aryl acylamidase solution prior to the start of the scan. This is due to the catalytic conversion of paracetamol to para-aminophenol by the enzyme.

Example 2 Steady-State Measurement in Buffer Solution In steady-state measurement, the current produced when a fixed potential was applied to the agitated solution was evaluated on the Y axis of a chart recorder using the X axis as the time base. After 2 minutes have passed until the system reaches equilibrium, the potential is +250 mV against SCE at 37 ° C.
Stable in. Agitation of the solution ensures that the layer of material adjacent to the electrode that is effective for oxidation is replenished, so that the current generated at the electrode is not attenuated by exhaustion of the reagent.

The stirred solution is 200 μl (1.5 units) of aryl acrylic amidase solution and 800 μl (pH 7) of buffer solution.
Including and Steady-state electrochemical measurements were performed under increasing amounts of paracetamol solution to generate a primary calibration curve for paracetamol. This is shown in FIG.

A similar primary calibration curve was prepared from a pH 7.5 buffer solution (potassium dihydrogen phosphate 2.18 g (BDH Analer) and dipotassium hydrogen phosphate 19.17 g (BDH Analer) dissolved in Milly Q water, The pH was adjusted to 7.5 and the final volume was set to 1.) This is shown in FIG.

When the pH of the incubation mixture was further raised to 8.0 (12.11 g of Trizmabase dissolved in Milly-Q water (manufactured by Sigma Chemical Co.), the pH was adjusted to 8 with 1M HCl, and the final volume was adjusted to 1 , An extremely inadequate response to paracetamol was observed, and a calibration curve shown in FIG. 6 that deviated from the straight line was generated at a paracetamol concentration of 0.5 mM or more.

A calibration curve for paracetamol obtained with buffers between pH 7.0 and 7.5 could be used in conjunction with the direct readings of unknown samples to determine paracetamol concentration.

Example 3 Steady state determination with 100% control serum Control serum (Monitrol IIE: Merz & Dade AG, Switzerland) was suspended in 3.5 ml Milly-Q water per vial (manufacturer's report). 70% of the volume listed in), then 143% mixed according to the manufacturer's report.
Control serum (final concentration) was obtained. Paracetamol (manufactured by Sigma Chemical Co.) was added to this control serum to give a final concentration of 4.
29 mM was obtained. Dilution of the above solution with 143% control serum gave a range of paracetamol concentrations in serum from 0 to 4.29 mM.

The agitated cell contained 700 μl of paracetamol solution in 143% control serum, 100 μl of 1M potassium phosphate buffer solution (pH 7.0), and 200 μl of aryl acylamidase solution (1.5 units) (agitated. The final concentration of serum in the cells performed was 100% equivalent). The steady state current was measured twice as described in Example 2 above. The calibration curve for paracetamol in 100% control serum is shown in FIG.

In a comparative study, paracetamol solutions at 143% control serum were analyzed using the standard paracetamol assay method [Cambridge Life Sciences (CLS)]. A good correlation was found between the measured steady-state current and the paracetamol concentration determined by the CLS method shown in FIG.

The particular advantage of Pseudomonas enzymes over Fusarium enzyme is that the latter is at ambient temperature and neutral pH (pH of whole serum is around 7.
2) works well. Such optimal conditions have facilitated the production of biosensors that can be used in any environment (eg operating room) without the need for special laboratory conditions.

Example 4 Enzyme Immobilization on Membrane All immobilized enzymes were mixed at various concentrations with a solution of cellulose acetate in acetone. The solution is then placed on the electrode surface and the acetone is evaporated (with or without a dry air stream),
The cellulose acetate membrane with the entrapped enzyme was left behind. Next, the fixed concentration of paracetamol was detected using this enzyme electrode (see FIG. 9).

The enzyme clearly retained its activity when immobilized in this way. Encapsulation of the enzyme did not significantly constrain its structure as it does not form a covalent bond with the immobilized membrane, which retained many of its natural activities.

The slow response times encountered with immobilized enzymes are likely due to enzyme activity (due to a small constraint on movement imposed by confinement) and restriction in product transport across the membrane.

The particular advantages of being able to immobilize an enzyme are: a) the ability to make a one-component device that senses paracetamol, b) the ability to reuse the enzyme, and c) the enzymatic properties of the sensor by immobilization. It can be changed to shorten the response time.

Various changes can be made within the scope of the present invention. For example, the present invention relates primarily to sensor electrodes, such as the electrodes specific to paracetamol, but the present invention also relates to the combination of electrodes with primary or permanent insertion means, such as needle probes. The invention also relates to such an electrode that is or can be associated with a signal or control device. The electrodes of the present invention enable the manufacture of improved macro-sensors for use in hospital analytical paracetamol or paracetamol derivative sensing machines.

The electrodes of the present invention are macroscale, simple for the surgeon, and can be incorporated into an inexpensive electronic digital reader. It will also be incorporated into an analysis kit.

The macro-sensor can be used in a device that can be slightly deformed to automatically take a blood sample from a finger, bring the sample into contact with the sensor, amplify the signal and digitally read it. In such applications, the sensor electrode can be composed of aryl acylamidase, stabilizer, buffer and dry graphite.

The present invention can be used in combination with the screen-printed electrodes described in the applicant's British patent application No. 8515884, "Amperometric sensor electrodes and method of making the same".

[Brief description of drawings]

Fig. 1 is a schematic diagram of a standard three-electrode device specified for all voltammetric measurements, Fig. 2 is a cyclic portammogram of p-aminophenol at a final concentration of 1 mM, and Fig. 3 is a paracetamol at a concentration of 1 mM. Explanatory drawing showing the influence of aryl acylamidase conversion on cyclic poltamogram, Fig. 4 is a calibration curve of paracetamol at pH 7.0, Fig. 5 is a calibration curve of paracetamol at pH 7.5, Fig. 6 Is a calibration curve of paracetamol at pH 8.0, FIG. 7 is a calibration curve of paracetamol at pH 7.0 in 100% control serum, and FIG. 8 is an analysis method and a display paracetamol analysis method of the present invention (Cambridge.・ A graph showing the results of comparison of analysis values with life science, Fig. 9 shows the amount of paracetamol immobilized using immobilized enzyme electrodes. It is a graph which shows the result.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location G01N 27/48 311 7363-2J

Claims (9)

[Claims] 1. A method for analyzing a liquid sample to determine the presence or content of N-acylated aromatic primary amines, comprising: (a) using the sample to provide a working electrode and an aromatic primary amine. Contacting with an enzyme of type EC3.5.1.13, which is an aryl acylamidase capable of catalyzing the hydrolysis of an acylated aromatic primary amine, (b) Balancing the charge on the oxidised electrode to transfer without the use of a mediator, (c) detecting the current as an indication of the presence or content of the N-acylated aromatic primary amine in the sample or A method for analyzing a liquid sample, which comprises measuring. 2. The analysis method according to claim 1, wherein the enzyme is obtained from bacteria. 3. The method according to claim 1, wherein the enzyme is screen-printed on a suitable substrate. 4. The analysis method according to claim 1, wherein the electrodes are screen-printed on a suitable substrate. 5. The first claim in which the sample is whole blood.
Analytical method described in paragraph. 6. A sensor electrode for analyzing the presence of paracetamol or a derivative of paracetamol by analyzing a liquid sample, the electrode hydrolyzing the paracetamol or derivative of paracetamol to provide an aromatic primary amine on its surface. It has an EC3.5.1.13 type enzyme, which is an aryl acylamidase that can catalyze amino acid, and oxidizes an aromatic primary amine to fish at a potential high enough to transfer charge to the electrode without the use of a mediator. A sensor electrode that can be matched. 7. The sensor electrode according to claim 6, wherein the enzyme is obtained from bacteria. 8. The sensor electrode according to claim 6, wherein the enzyme is screen-printed on a suitable substrate. 9. A sensor electrode according to claim 6, wherein the electrode is screen-printed on a suitable substrate.